Ceramides and derivatives thereof, are of great commercial potential in cosmetics and pharmaceuticals such as hair and skin care products (Zysman, A. et al. European Patent Application 420,722).
Ceramides are a class of polar lipids (sphingolipids) endogenous to the epidermis. Ceramides play a major role in the water-retaining properties of the epidermis. It has been found that topical applications of ceramide- and pseudo- ceramide- containing compositions are effective in restoring the water content of dry skin and may be effective in relieving atopic eczema (Kerscher, M. et al. (1991) Eur. J. Dermatol., 1, 39-43; Imokawa, G. et al. (1989) J. Soc. Cosmet. Chem., 40, 273-285).
Lysosphingolipids are sphingolipid breakdown products which lack the amide-linked fatty acyl group at the 2-position of the sphingoid base (Hannun, Y. A. and Bell, R. M. (1989) Science 243, 500-507).
In current practice, ceramides are primarily obtained via extraction and isolation from animal epidermal tissues, usually from bovine or porcine epidermal tissue. Obviously, this is a rather costly process on an industrial scale. Moreover, it has been found that these materials are potentially unsafe due to the possible presence of bovine spongiform encephelatis (BSE) in bovine tissue.
Various chemical methods have been published describing the synthesis of ceramides. However, these methods often have the disadvantage of lacking the proper stereochemistry of the thus-produced end-products. Since sphingolipids often have multiple chiral centers, it would be advantageous to employ synthesis methods which reliably provide only a desired stereoisomer, in order to obtain a product which more closely resembles the compound as it appears in nature. Moreover, these chemical synthetic methods may also leave amounts of undesired residual chemical reactants in the final products.
The use of enzymes in chemical synthesis to produce amides in organic solvent has been described (Zaks, A. and Klibanov, A. M. (1985) Proc. Natl. Acad. Sci. U.S.A., 82, 3192-3196). The enzymes most employed for this purpose are lipases (triacylglyceride ester hydrolases; EC 3.1.1.3) and esterases.
Margolin, A. L. and Klibanov, A. M. ((1987) J. Am. Chem. Soc., 109, 3802-3804) studied lipase-catalyzed peptide synthesis in anhydrous organic solvents. Porcine pancreatic lipases (PPL) and mold lipases were found to catalyze the synthesis of peptide bonds in toluene and tetrahydrofuran (THF). Despite the author's assertion that mold lipases have an efficiency comparable to that of PPL in peptide synthesis, we have found that mold lipases were unsuccessful in catalyzing the amidation of lysosphingolipids (a class of amino alcohols) in the synthesis of ceramides.
Bistline, R. G. et al ((1991) JAOCS, 68, 95-98) studied the lipase catalyzed formation of fatty amides. It was found that the three lipase preparations studied showed different degrees of activity and selectivity where hexane was used as the organic solvent. It was also acknowledged that the use of other solvents may produce different results.
Djeghaba, Z. et al ((1991) Tetrahedron Lett., 32, 761-762) reported that, in the enzymatic acylation of amides in ethyl butyrate, a Candida lipase SP 382 was more efficient than lipases from Candida rugosa, porcine pancreatic lipase, Pseudomonas lipase and horse-liver acetonic powder. This further demonstrates the enzyme- and solvent- dependent factors which affect lipase-catalyzed amidation reactions.
The use of lipases on reactants having multiple functional groups such as amino alcohols have also been reported. The product formation was found to depend largely on the type of lipase used and the solvent in which the reaction was performed. The results found in these articles clearly demonstrate the unpredictability of the use of various enzymes and solvent systems in the attempt to produce desired acylation products.
Montet, D. et al ((1989) Revue Francaise des Corps Gras, 36, 79-83) studied the acylation of aminopropanols using Mucor miehei lipase in organic solvent. The selectivity of the acylation (N- or O- acylation) was found to be influenced by the type of solvent used in the reaction medium. This particular lipase, however, failed to produce a positive result when employed in amidation reactions of lysosphingolipids in the synthesis of ceramides.
Graille, J. et al (European Patent Application 298,796) disclose the synthesis of the fatty acid amide by enzymatic catalysis. The specification teaches that lipases, acylases, peptidases, proteinases and amidases all may be used in the disclosed method. Only Mucor miehei lipase was actually exemplified. We have found that this lipases is ineffective in the amidation of lysosphingolipids.
Montet, D. et al ((1989) Fat Sci. Technol., 91, 14-18) have also published another study of the effect of water activity on the synthesis of N-lauryloleylamide using Mucor miehei lipase in various solvents. As mentioned above, this lipase failed to produce a positive result when used for the synthesis of ceramides.
Gotor, V. et al (1988) J. Chem. Soc., Chem. Commun., 957-958) described the enantioselective acylation of amino alcohols by porcine pancreatic lipase in organic solvents. Ethyl acetate was found to provide enantioselective results. However, other solvents such as chloroform, benzene and THF did not give satisfactory results.
Chinsky, N. et al ((1989) J. Am. Chem. Soc., 111, 386-388) described the chemoselective enzymatic mono-acylation of bi-functional compounds. Surprisingly, O-acylation products predominated when certain amino alcohols were acylated via the use of porcine pancreatic and Pseudomonas lipases in tert-amyl alcohol.
Gagin, V. I. C. ((1991) Biofutur, 40-46) disclosed the successful acylation of amino sugars using Mucor miehei lipase in tert-amyl alcohol. These conditions were found to be unsuitable for the amidation of lysosphingolipids.
The foregoing descriptions of the publications forming the state of the art at the present time show the unpredictable nature of the synthesis of amides using various enzymes and solvent systems.
Until now, a reliable method of producing ceramides using enzymatic activity has not been described.